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Abstract:

The present invention relates to an analysis method used to quantitatively
compare the expression levels of the same gene from different sources.
The method of the present invention can be used to quantitatively compare
the gene expression level difference of the same gene of tissues or cells
from different sources, making use of the quantitative characteristics of
bioluminescent assay and the principle of adding different
deoxyribonucleic acids (dNTP) one by one. The concrete steps are: reverse
transcript the messenger ribonucleic acids (mRNA) from different sources
into cDNA, and label a segment of source specific sequence in cDNA from
each source; mix the labeled cDNA of different sources into one tube and
use it as the substrate of polymerase chain reaction (PCR); PCR
amplification is performed using the same common primer and a
gene-specific primer; Detect the base sequence by bioluminescent assay,
wherein the base type represents the different gene source, and the
signal intensity of each base represents the gene expression level from
each source. This method has a significant meaning for the screening of
disease-related genes, clinical early diagnosis and the preparation of
specific medicine for the treatment of disease.

Claims:

1. A comparing method for expression amount of the same gene from
different sources by base sequence measurement comprising:(a) use DNA
sequence labeling method to make the reverse transcript complementary DNA
(cDNA) of mRNA from different sources contain a segment of
source-specific sequence;(b) mix the labeled cDNA of different sources
into one tube and use it as the substrate of polymerase chain reaction
(PCR); PCR amplification is performed using the same common primer and a
gene-specific primer;(c) Detect the base sequence of the above PCR
amplified products corresponding to the gene sources by bioluminescent
assay, wherein the base type represents the gene source, and the signal
intensity of each base represents the gene expression level from each
source.

2. The comparing method of claim 1, wherein the said different sources
mean different tissues or cells.

3. The comparing method of claim 1, wherein the said DNA sequence labeling
method means that restriction endonuclease is used to prepare double
stranded cDNAs from different sources into fragments of proper length
through enzymolysis; and then ligate the fragments with DNA adapters of
different sequences--cDNA from different sources is ligated with
different DNA adapters.

4. The comparing method of claim 3, said DNA adapter contains the sequence
complementing to the cuts of the restriction endonuclease in claim 3, and
is composed of two single strand DNAs that are not completely
complementary to each other; and the adapter can ligate with double
stranded cDNA enzymatic fragments in the action of ligases.

5. The comparing method of claim 4, wherein one of said two single strand
DNAs that are not completely complementary to each other contains a
segment of gene source-pecific sequence, and a base sequence that does
not change with gene sources is between this sequence and the 5' terminus
of this strand, and this base sequence is not complementary to the 3'
terminus of another strand.

6. The comparing method of claim 1, wherein the said DNA sequence labeling
method means that reverse transcript mRNAs from different sources with
primers of different sequences respectively, making cDNAs from different
sources labeled with different DNA fragments.

7. The comparing method of claim 6, wherein the said primers of different
sequences means that the primer's 3' terminus is composed of multiple
thymines, and there is a gene source-specific sequence between the 3'
terminus and the 5' terminus, and a base sequence that does not change
with gene sources is between this sequence and the 5' terminus of this
strand.

8. The comparing method of claim 1, wherein the said common primer means
that the primer's sequence is partly or completely similar with the base
sequence (as mentioned in claim 5 and claim 7) that does not change with
gene sources.

9. The comparing method of claim 1, wherein said bioluminescent assay
means the method that quantitatively assay the pyrophosphate (ppi)
produced by extension reaction.

10. The comparing method of claim 9, wherein said extension reaction
means: use the PCR amplified product of claim [1] or its single strand
product as template, add the sequencing primers to anneal, then orderly
add dNTP, or ddNTP or their analogues, in the action of DNA polymerase,
when the added dNTP or ddNTP or their analogues complementary to the
template, the polymerization occurs.

Description:

[0002]The present invention relates to a method used to quantitatively
compare the relative expression level of the same gene of tissues or
cells from different sources. Specifically, it is a base sequencing
method for the determination of the relative content of each DNA fragment
in the mixture of DNA fragments labeled by different base sequences and
all the determinations can be carried out at the same time.

BACKGROUND OF THE INVENTION

[0003]With the progress in molecular biology and analytical apparatus, the
sequencing work of human genome project (HGP) has already been finished.
As the structure of the whole human genome is clarified, the next step is
to analyze gene functions coded in genomes.sup.[1], gene function
analysis includes the understanding the distribution of the gene
transcription products mRNA and the quantity, distribution and function
of proteins (the translated products of mRNA) in a cell or different
organs in a body. We can look for and find the disease-related genes by
comparing the gene expression levels between healthy persons and
patients, and further make them used in clinical early diagnosis.sup.[2].
In drug screening process, the target of drug can be found by detecting
the relative gene levels of the administration group and the untreated
group, and further look for and prepare the specific medicine for the
treatment of disease.sup.[3]. Therefore, the differential analysis of
gene expression level has become one of the main tasks of the
"post-sequencing age". The developed countries have invested a lot of
material resources and money to rank top in this field and further
monopoly the technology. At present the major analysis methods for the
gene expression level comparison are: SAGE method.sup.[4], RT-PCR
(reverse transcription-polymerase chain reaction) method.sup.[5] and
microarray (gene chip).sup.[6], etc. But these methods still have some
drawbacks: only the gene expression levels of two individuals can be
compared at a time; the prices of apparatus are very high; the operation
is complex and the quantitative characteristics are bad, etc. For
example, in the case of SAGE method, detection is very tedious and there
are too many steps so it is hard to control, in addition, the cost is
also very high, all these lead to its small popularity. RT-PCR method
needs special apparatus and internal standard, its detection is also
tedious and the repeatability is bad. Microarray is a high-throughput
detection method, although the detection amount is large and multiple
genes can be detected on one chip at the same time, samples should be
labeled by fluorescent dyes, the sensitivity is bad, apparatus are
expensive and the data processing is complex, therefore, it is hard to
accurately compare the gene expression levels of a given gene from
different sources.

[0004]It is a new developed method to determine base sequence by
bioluminescence technology.sup.[7-8]. This method is convenient and
rapid, and has the advantages of cheap apparatus, low cost and easy to
realize automation. But this method is limited to analyze the mutation
and polymorphism of genes for it can only detect 10 to 30 base
sequences.sup.[9].

SUMMARY OF THE INVENTION

[0005]The purpose of the present invention is to study a method for the
assay of relative gene expression level of a given gene from different
sources by base sequencing technology. That's to say, how to detect the
relative gene expression levels by detecting several base sequences, and
establish a convenient method with high sensitivity, accurate
quantification and low cost, which can be used in clinical diagnosis.

[0006]Technological solutions of the invention are as below, and the
detection principle is shown in FIG. 1:

[0007](1) Label a Given Gene from Different Sources by Base Sequencing
method. [0008]This can be realized by two methods. The first method is
DNA adapter labeling method. That's to say, first extract the total RNA
or mRNA of tissues or cells from different sources, and reverse
transcript them into double stranded cDNA, and then use restriction
endonuclease to cut the cDNA from each source into DNA fragments of
different lengths; ligate the cDNA enzymatic products from each source
with DNA adapters that can differentiate the sources, making the cDNA of
each source labeled with DNA adapters of different sequences. DNA
adapters are composed of two single strand DNA that are not completely
complementary to each other, and its structure is shown in FIG. 2. That's
to say, one of its ends contains sequence 1 that is complementary to the
cut of the above restriction endonuclease; and it ligate with double
stranded cDNA enzymatic fragments in the action of ligases; the other end
is "Y" structure, which is made up of a pair base sequences 2 and 3 that
are not complementary to each other. Sequence 3 is also can be designed
to the one complementary to sequence 2, but in this case, the 3' terminus
of sequence 3 must be properly modified to make it won't perform
extension reaction in the action of polymerase. Sequence 2 contains a
gene source-specific sequence 4, and sequence 5 that won't change with
gene sources is between this sequence and the 5' terminus of this strand.
Different gene source-specific DNA adapters can be designed into such
state that the base sequence is only different at sequence 4, but the
type and number of bases forming this sequence are the same. [0009]The
second method is reverse transcription primer labeling method. That's to
say, first extract the total RNA or mRNA of tissues or cells from
different sources, and reverse transcript them into cDNA with primers of
different sequences, making cDNA from each source labeled with DNA
fragments of different sequences. The structure of reverse transcription
primer is shown in FIG. 3. Its 3' terminus (sequence 1 in the Figure) is
composed of multiple thymines, and a gene source-specific sequence 2 is
between the 3' terminus and the 5' terminus, and a base sequence 3 that
does not change with gene sources is between this sequence 2 and the 5'
terminus of this strand. Different gene source-specific reverse
transcription primers can be designed into such state that the base
sequence is only different at sequence 2, but the type and number of
bases forming this sequence are the same.

[0010](2) PCR Amplify the Same Gene from Different Sources on an Equal
Proportion Basis. [0011]Usually, the expression level of the target
gene extracted from tissues is small and it can be detected only by PCR
amplification. One of the key technologies of this patent is how to
amplify the above labeled gene from different sources in a monotube on an
equal proportion basis. First we should design a gene specific primer
(GSP) according to the sequence of the target gene; meanwhile design
another common primer (CP), and its sequence is the same as the 5
sequence (when labeled with DNA adapters) in FIG. 2 or the sequence 3
(when labeled with revere transcription primer) in FIG. 3. In the
condition that primers CP and GSP are present, if some source contains
the target gene then GSP will first anneal with it and extension reaction
occurs, the primer CP anneals with the extension product and extension
occurs; if there is no extension product of GSP, then primer CP won't
extend. A pair of primers CP and GSP is used to amplify the same gene
fragment from different sources and the Tm values of the amplified
products are totally the same (the length and the base species are the
same), so the PCR amplification can be ensured on an equal proportion
basis. [0012]If the arm 2 and arm 3 in FIG. 2 are complementary to each
other, then the arm 3 will extend in the action of DNA polymerase,
producing the template for CP annealing. Therefore, the PCR amplification
of the same gene from different sources cannot be realized on an equal
proportion basis.

[0013](3) The Sequencing of the Amplified Products of the Same Gene from
Different Sources. [0014]At present the commonly used sequencing
reaction is based on the principle of gel electrophoresis, which is
qualitative detection, and the quantitative characteristic is bad;
furthermore, it cannot detect the base sequence following the primer,
that's to say, it cannot detect the sequences of the first 50 bases.
Bioluminescent assay is based on PPi detection. For example,
pyrosequencing is a method for sequencing by orderly adding each dNTP in
cycle, which can not only directly detect the base sequence following the
primer, the quantitative characteristic is also very good, and the number
of the repetitive bases can be determined by measuring the peak height.
The present invention detects the sequences of the amplified products of
the same gene from different sources by sequencing method based on PPi.
The base type in sequence can be used to differentiate the gene source,
and the peak intensity can be used to judge the gene expression level
difference of different sources. Gene expression level analysis is the
important content of genomics research. The purpose of the present
invention is to apply the sequencing technology to the comparative assay
of gene expression level difference. Compared with the present
technology, the innovation of the invention is: the expression level
difference of the same gene from different individuals can be detected by
only one assay, and additional detection cost is not required. Easy to be
instrumented, laser, gel, fluorescence labeling, and electrophoresis are
not required. This inventive method has a wide application prospect for
its advantages of high sensitivity, good quantification, low price and
simple operation. This method has a significant meaning for the screening
of disease-related genes, clinical early diagnosis and the preparation of
specific medicine for the treatment of disease. And it also can be used
to study the expression of the relative genes when human beings, animals
or cells are in the treatment of drugs or other methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is the principle chart of detecting the difference of gene
expression level between various sources of the present invention.

[0018]FIG. 4 is the sequencing result when using DNA adapters to label the
P53 genes in human brain cancer tissue, normal tissues and liver cancer
tissue.

[0019]FIG. 5 is the sequencing result when using reverse transcription
primers to label the P53 genes in human liver cancer cell and human
bladder cancer cell.

DETAIL DESCRIPTION OF THE INVENTION

[0020]Concrete examples are used to illustrate the above method, the main
experimental steps are as below:

[0021](1) Label the same gene from different sources. Respectively extract
the total RNA or mRNA of relative tissues or cells from each individual
and detect its concentration. Then according to DNA adapter labeling
method or reverse transcription primer labeling method, make the cDNA
from each source labeled with DNA adapters of different sequences or DNA
fragments of different sequences. When labeling with DNA adapter labeling
method, first reverse transcript the mRNA into double stranded cDNA; cut
the cDNA into fragments of a certain length by restriction endonuclease
(Mob I) that can identify the sequences of the four bases. Respectively
ligate these fragments with the DNA adapters containing gene
source-specific sequences. Then mix cDNA fragments of different sources
that are labeled with DNA adapters, and use the mixture as the template
of PCR amplification reaction. When labeling with reverse transcription
primer labeling method, respectively reverse transcript mRNA into cDNA
with the reverse transcription primers that are corresponding to each
source, mix after purification, and use the mixture as the template of
PCR amplification reaction.

[0022](2) PCR amplification. Perform PCR amplification reaction on the DNA
template in (1) using a common primer (CP) that is not relevant to gene
source and a gene specific primer (GSP). Because a pair of primers CP and
GSP are used to amplify the same gene from multiple sources, the relative
proportion of the given gene in the each source is fixed in the
amplification process, that's to say, the amplification is performed on
an equal proportion bases, and the proportion won't change with the
increase of amplification times. If the expression level difference of
multiple genes from the above different sources needs detecting,
different GSP needs to be added to perform the amplification.

[0023](3) Quantitatively detect the amplification products of the gene
fragments from different sources at the same time. After the single
strand is prepared from the amplified PCR product by biomicrosphere
technique or restriction enzyme digestion, add the common primer
complementary to the template sequence to anneal. Or directly purify the
PCR products, and then add the common primer complementary to the
template sequence to anneal. Then detect the sequences of two bases by
bioluminescence method, namely respectively add dNTP or ddNTP
corresponding to the gene sources to the solution containing substrate.
If the added dNTP or ddNTP is complementary to the template, then PPi
will be released. PPi is converted into ATP rapidly in the action of
enzyme, and ATP reacts with luciferin in the action of luciferase to
produce light signal. In the result, the base sequence represents the
different gene source, and the signal intensity represents the gene
expression level of each source. According to the gene expression level
difference of each individual, we can quickly judge the function of genes
and find the disease-related functional genes.

[0025]This example detects the expression levels of P53 genes of the
tissues from three different sources by DNA adapter labeling method.
First, design three different DNA adapters, respectively ligate them with
the cDNA fragments digested by restriction endonuclease, and then mix to
perform PCR amplification.

[0026]1. The Preparation of cDNA Sample.

[0027](1) The extraction of total RNA: respectively get 0.1 g human normal
tissue, brain cancer tissue and liver cancer tissue, add 1 ml Trizol to
the Tissuelyser to grind, extract total RNA according to the operation in
the Trizol instruction. Identify it by electrophoresis, if the 28 s and
the 18 s bands are complete and have no degradation, detect its
concentration by ultraviolet absorption method, and then regulate its
final concentration to 1 μg/μl with sterile DEPC-H2O.

[0031](1) Enzyme digestion reaction: add 10 μl double stranded cDNA, 2
μl buffer solution of 10 times' concentration, 1 μl Mbo I TaKaRa
endonuclease (10 U/μl), 7 μl distilled water for sterilization, the
total volume of the reaction system is 20 μl. Place the mixture in
37° C. water bath and react for 2 h, and then place it at
70° C. for 10 min to inactivate the Mbo I enzyme. The feature of
the Mbo I endonuclease is that it can identify the 5'→3' GATC
order in DNA, and cut it to form the GATC adhesive end with the 5'
terminus bumps.

[0032](2) Ligation reaction: get equal volume of endonuclease reaction
solutions of different sources; respectively ligate them with 3 different
DNA adapters. One strand adp-4 of the three DNA adapters is the same,
another strand contains four gene source-specific bases, and the four
bases only have different sequence, all of them are composed of c, t, g,
and c. Their sequences are: adp-1: 5'-ccc cac ttc ttg ttc tct cat gtca cg
cat cac tcg-3', adp-2: 5'-ccc cac ttc ttg ttc tct cat ctga cg cat cac
tcg-3'; adp-3: 5'-ccc cac ttc ttg ttc tct cat atcg cg cat cac tcg-3';
adp-4: 5'-gat ccg agt gat gcg cta ag-3'. The parts having underlines and
italic are gene source-specific bases. adp-1 and adp-4 form a DNA adapter
1, adp-2 and adp-4 form a DNA adapter 2, adp-3 and adp-4 form a DNA
adapter 3. All of the adapters have the structure with the 5' terminus
bumps the four bases GATC. DNA adapters 1, 2 and 3 are respectively used
to label the P53 genes in human normal tissue, brain cancer tissue and
liver cancer tissue. Get 1 μl enzyme digestion solution, add 2 μl
two single strands (10 p mol/L) that form the adapters (2 μl each), 2
μl 10×T4 DNA ligase Buffer and 11 μl distilled water for
sterilization, place the mixture at 70° C. for 10 min, and then
cool down the temperature to 16° C. at the rate of 0.2°
C./s, add 2 μl T4 DNA ligase (4 U/μl) and react for 2 h.

[0035](2) The preparation of single strand: get 25 μl M280 beads and
wash them according to the requirements of operation instruction.
Dissolve in 50 μl 2× B&W Buffer (wash buffer), add equal volume
of PCR product to react 30 min, shake slightly in the reaction process so
as to make the beads in a suspension state. Fasten the beads with magnet
and desert the supernatant, add 20 μl NaOH solution (0.1 mol/L) after
washing the beads 2-3 times with 1× B&W Buffer, and then react for
5 min. Extract the supernatant to another tube and regulate the pH value
to 6˜7 with diluted hydrochloric acid, and then store it in
refrigeration. The solid phase beads are dissolved in the 1× B&W
Buffer to store after being washed and leave it to use when sequencing.

[0036]4. Compare the Relative Expression Level of the Same Gene from
Different Sources by Base Sequence Determination Method.

[0037]Prepare the solutions that contain 25 mM Mg2+ and 5 mM Tris(pH
7.7) from the above single strand DNA sample (the biomicrosphere in step
3), and respectively add 5 pmol CP into each solution, heat the solutions
at 70° C. for 10 min and then naturally cool them down to room
temperature. Get 1˜5 μl solution and add it into sequencing
detection standard mixed solution of 100 μl, and then orderly add dNTP
to perform sequencing reaction.

[0040]For DNA adapters 1, 2 and 3 are respectively used to label the P53
genes in human normal tissue, brain cancer tissue and liver cancer
tissue, when adding dGTP, the obtained signal intensity represents the
gene expression level from human normal tissue; when adding dCTP, the
obtained signal intensity represents the gene expression level from human
brain cancer tissue; when adding dATPaS (the analogue of dATP), the
obtained signal intensity represents the gene expression level from human
liver cancer tissue. The sequencing result is shown in FIG. 4. The first
base "C" of the sequence in the Figure is from the DNA adapter 2,
representing the expression level A1 of the P53 gene in human brain
cancer tissue; the second base "G" is from DNA adapter 1, representing
the expression level A2 of the P53 gene in human normal tissue; the third
base "A" is from DNA adapter 3, representing the expression level A3 of
the P53 gene in human liver cancer tissue. The ratio of peak heights of
the three base sequences represents the expression level difference of
the P53 gene in the three sources. The two times' detection results
(A1:A2:A3) are: 28.20:24.9:46.9 and 28.1:22.4:49.5, average ratio
(A1:A2:A3) is: 28.15:23.65:48.2.

[0042]This embodiment mainly use reverse transcription primer labeling
method to detect the expression level difference of P53 gene in human
liver cancer cell and bladder cancer cell. That's to say, use primers of
different sequences to respectively reverse transcript mRNA from
different sources, making the cDNA from different sources labeled with
DNA fragments of different sequences. And compare the result with the
RT-PCR detection result.

[0047]Perform PCR amplification and prepare single strand according to the
method in [Embodiment 1]. Wherein the CP and gene specific primer are the
same as that of [Embodiment 1].

[0048]3. Comparatively Assay the Gene Expression Level of the Same Gene
from Different Sources by Base Sequence Determination Method.

[0049]Prepare the solutions that contain 25 mM Mg2+ and 5 mM Tris(pH
7.7) from the above single strand DNA sample, and respectively add 5
pamol CP into each solution, heat the solutions at 70° C. for 10
min and then naturally cool them down to room temperature. Get 1˜5
ml solution and add it into sequencing detection standard mixed solution
of 100 ml, and then orderly add dNTP to perform sequencing reaction.

[0052]The sequencing result is shown in FIG. 5. The first base "C" of the
sequence in the Figure is from the reverse transcription primer P-1,
representing the gene expression level A1 of human liver cancer cell; the
second base "G" is from the reverse transcription primer P-2,
representing the gene expression level A2 of human bladder cancer cell.
The ratio of peak heights of the two base sequences represents the
expression level difference of the P53 gene in the two sources. The two
times' detection results (A1: A2) are: 82.9:17.1, 87.4:12.6, 84.2:15.8,
89.5:10.5, average value is: 86:14=6.14:1, standard deviation of the
detection is 3.0:3.0.

[0053]The expression levels of P53 gene in liver cancer cell and bladder
cancer cell by RT-PCR method are 126359 copies/μl and 22093/μl, the
ratio is 5.72:1. Compare the detection result of the two methods; the
relative average deviation is less then 2%, which indicates that the
detection result of the method in present invention is more accurate.